7330
Biochemistry 1992, 31, 7330-7340
Purification and Characterization of a Novel Growth Factor from Human Breast Cancer Cells+ Ruth Lupu,’ Anton Wellstein, James Sheridan, Bruce W. Ennis, Gerhard Zugmaier, Deborah Katz, Marc E. Lippman, and Robert B. Dickson The Vincent T. Lombardi Cancer Research Center, Georgetown University Medical Center, 3800 Reservoir Road, Washington, DC 20007 Received April 8, 1992
ABSTRACT: We have purified and characterized a novel 30” glycoprotein (gp30) with TGFa-like properties secreted from the estrogen receptor negative breast cancer cell line MDA-MB-23 1. This factor was immunoprecipitated by an anti-TGFa polyclonal antibody and also had TGFa-like biological activity, as assayed by EGF radioreceptor assay and anchorage-independent assays. In addition, the novel growth factor stimulated phosphorylation of the EGF receptor and erbB-2 receptor. However, the novel growth factor, unlike EGF and TGFa, bound to heparin-Sepharose. Purification of gp30 was obtained to apparent homogeneity by heparin affinity chromatography and subsequent reversed-phase chromatography. Tunicamycin treatment in vivo or N-glycanase deglycosylation in vitro revealed a putative precursor of approximately 22 kDa molecular mass in contrast to the “normal” 16-kDa precursor species for TGFa. In vitro translation of total mRNA from MDA-MB-231 cells confirmed the size of the putative precursor. Biochemical characterization of gp30 was begun by V8 protease digestion of the deglycosylated polypeptide and the translated products. Peptide mapping of V8-digested, immunoprecipitated material suggests that the amino acid sequence of this unique protein is distinct from mature TGFa and not the result of a posttranslational modification of the precursor. We conclude that this TGFa-like (gp30) polypeptide is a novel growth factor with agonistic activity for both EGF and erbB-2 receptors.
Transforming growth factors belong to a family of heatand acid-stable polypeptides which allow cells to assume a transformed morphology and form progressively growing colonies in anchorage-independent growth assays (DeLarco & Todaro, 1978; Moses et al., 1981; Ozanne et al., 1980; Roberts et al., 1980). Transforming growth factor type a (TGFa)’ is a polypeptidewhose expressionin rodent fibroblasts is selectively induced after cellular transformation by a variety of oncogenes. TGFa was first purified to homogeneity from conditioned media from the human melanoma A2058 cell line (Marquardt et al., 1982). It has been isolated from various solid tumors (Nickel1 et al., 1983)and from conditionedmedia from a variety of human cancer cell lines including breast cancer cell lines (Todaro et al., 1980; Zwiebel et al., 1982). TGFa is a structural and functional analogueof epidermal growth factor (EGF). TGFa and EGF are small polypeptides (molecular mass of 6 kDa) encoded by separate genes (Salomon et al., 1984; Gray et al., 1983), and they share approximately 40% of their amino acid sequence (Derynck et al., 1984; Massague, 1983; Marquardt et al., 1983). Despite differencesin primary structure, TGFa and EGF have similar secondarystructure and bind with nearly equal affinity to the human EGF receptor (Marquadart et al., 1982). Systemsused for the analysis of TGFa activityinclude A43 1 human carcinoa cells, which overexpress the EGF This work was supportedby American Cancer Society GrantsCD459 and BE-61956. * Author to whom correspondence should be addressed. Telephone: (202) 681-3795. Fax: (202) 681-6402. Abbreviations: TGFa, transforming growth factor a;TGFa-like, transforminggrowth factor a like;EGF, epidermalgrowth factor; HPLC, high-performanceliquid chromatography;FPLC, fast protein liquid chromatography; NRK, normal rat kidney; HEPES,4-(2-hydroxyethyl)-lpiperazineethanesulfonicacid; SDS, sodiumdodecyl sulfate;NRS, normal rabbit serum; R399, rabbit antirecombinant TGFa; RRA, radioreceptor assay; RIA, radioimmunoassay.
0006-2960/92/043 1-7330$03.00/0
receptor and NRK-49F rat fibroblasts, both of which require TGFa for anchorage-independent growth (Marquardt et al., 1984). Mature TGFa is a single-chain polypeptideof 50 amino acids, and is initially synthesized as a transmembrane, palmitoylated species. Its 16-18-kDa precursor protein is biologically active and is equipotent with the mature polypeptide (Massague, 1985; Ignotz et al., 1986). TGFa is a mitogenic polypeptide which is also produced by a variety of retrovirally-, chemically-, or oncogene-transformed human and rodent cell lines [reviewed by Sporn et al. (1985)l. In addition, it is present in human mammary tumor tissue and cell lines (Solomon et al., 1984; Derynck et al., 1987a,b) and has recently been demonstrated in normal human keratinocytes (Coffey et al., 1987). There have been numerous other reports of a bioactive higher molecular weight form of TGFa found in the supernatant of cultured rat hepatocellular carcinoma cells (Luetteke et al., 1988), extracts of human milk (Zwiebel et al., 1986), alveolar macrophages (Madtes et al., 1988),andurine (Hudginsetal., 1987). Thesignificance of the presence of TGFa in these tissues and fluids is unknown. Two recent reports have described new members of the EGF/ TGFa family: the amphiregulin gene derived from MCF-7 human breast cancer cells treated with tetradecanoylphorbol acetate (Shoyab et al., 1988) and another molecule derived from human malignant gliomas (Rutka et al., 1989). The range and regulation of expression of these two genes are relatively unknown. In many cases, high molecular weight forms of TGFa constitute the predominant TGFa activity, and their estimated sizes are equal to or exceed the molecular weight predicted for the transmembrane precursor. However, their precise structural relationship with the TGFa precursor remains undefined. The TGFa cDNA sequence encoding the 160 amino acid precursor contains potential sites for posttranslational modification of the TGFa protein in the human (Derynck et al., 0 1992 American Chemical Society
TGFa-like Protein: Purification and Characterization 1984) and rat (Lee et al., 1985), largely via glycosylation. The presence of a hydrophobic sequence in the carboxyl terminus of the protein suggests that the precursor is a transmembrane protein from which mature TGFa is released by the action of a specific protease. The release of mature TGFa from its 16-18-kDa precursor may occur in vivo by the action of an elastase-like enzyme, which cleaves at sites containing clusters of apolar amino acids, in particular near valine and alanine residues (Geneste et al., 1969). Elastase can cleave the 16-18-kDa TGFa precursor from Rat-FeSrV cells into a 6-kDa product, suggesting a high degree of specificity (Ignotz et al., 1986). A TGFa cDNA probe encoding the 160 amino acid precursor recognizes a 4.8-kb mRNA species in a variety of breast cancer cell lines such as MCF-7, ZR 75B, MDA-MB23 1, and T47D, but not HS578T, by Northern blot analysis (Derynck et al., 1984, 1985, 1987a,b). MDA-MB-231 cells secrete mature 6-kDa TGFa and also a large size TGFa-like polypeptide detected by EGF receptor binding of fractionated conditioned media (Bates et al., 1988). Because human breast cancer cell lines and human breast tumors contain EGFITGFa receptors, an autocrine pathway can be proposed for the TGFa. According to this hypothesis, cells secrete mitogenic polypeptides which act through their own cell-surface receptors to stimulate growth. In addition, an estrogen-regulated autocrine or paracrine growth stimulatory pathway involving TGFa in MCF-7 cells was proposed (Bates et al., 1988). Conditioned media from confluent cultures of MDA-MB-231 human breast cancer cells also stimulated proliferation of MCF-7 cells in a dose-dependent and saturable manner. The high levels of expression and secretion of a high molecular weight TGFa-related polypeptide by MDA-MB-231 cells (Bates et al., 1988) provided a suitable source for its purification and characterization. Heparin affinity chromatography, which has been used for the isolation of endothelial cell growth factors (Shing et al., 1984; Klagsbrun et al., 1984; Gospodarowicz et al., 1984; Maciag et al., 1984) and has recently been shown to bind a high molecular weight form of an uncharacterized TGFalike from activated alveolar macrophage (Madtes et al., 1988) and an EGF-like heparin binding polypeptide (Higashiyama et al., 1991), was used for the isolation of the high molecular weight TGFa-like polypeptidefrom MDA-MB-231 cells. The human breast cancer TGFa-like growth factor was isolated from conditioned media from MDA-MB-231 cells, a highly invasive and estrogen receptor-negativebreast cancer cell line. We describe a novel isolation and characterization of a unique high molecular weight growth factor. This growth factor is unique as determined by peptide mapping both of purified gp30 and of its in vitro translated p22 precursor. The novel growth factor has also been shown to activate tyrosine phosphorylation of the erbB-2 receptor (Lupu et al., 1990).
MATERIALS AND METHODS Cell Lines. Cells from the following sources were used in these studies: MDA-MB-231 and NRK clone 49F fibroblasts were obtained from the American Type Culture Collection (Rockville, MD). Hs578T cells were received from Helene Smith (Hackett et al., 1977). A431 cells were donated by Ira Pastan (Giard et al., 1973). H8 cells, a human TGFa-transfected MCF-7 breast cancer cell line, were kindly provided by Francis G. Kern (Clarke et al., 1989). Carcinogenimmortalized normal mammary epithelial cell subline 184AlN4 and its SV40-transfected derivative 184AlN4T were kindly provided by Robin Clark (Clark et al., 1988).
Biochemistry, Vol. 31, No. 32, 1992 7331
Rat-FeSrV-transfected cells were kindly provided by Joan Massague (Ignotz et al., 1986). All cell lines were propagated in improved modified Eagle’s medium (IMEM; G i b , Grand Island, NY) supplemented with 10% fetal bovineserum (FBS, Gibco). Conditioned Media Preparation, Collection, and Concentration. Conditioned media collections were carried out as previously described (Bates et al., 1986). The media were concentrated 100-foldin an Amicon ultrafiltration cell (YM5 membrane) (Amicon, Danvers, MA). Once clarified and concentrated, the media were stored at -20 OC while consecutive collections were made during the following days. The concentrated media were dialyzed using Spectrapor 3 tubing (Spectral Medical Industries, Los Angeles, CA) against 100 volumes of 0.1 M acetic acid over a 2-day period at 4 OC. The material that precipitated during dialysis was removed by centrifugation at 4000 rpm for 30 min at 4 OC; protease inhibitors were added. Metabolic Labeling and Immunoprecipitation. Cells were grown to 80% confluence in IMEM. Cell monolayers were washed 3 times with PBS and incubated for 2 h in serum-free IMEM which lacked methionine and cysteine and was supplemented with glutamine (2.9 g/L) (Biofluids, Rockville, MD). This medium was then removed and replaced with serum-free IMEM without methionine and cysteine containing 2.5 mCi/mL [35S]cysteineand methionine (Amersham, Arlington Heights, IL; 1175 Ci/mmol). A total of 2.5 mL of this meidum was used for a 5-cmdish. The medium washarvestedfromthecultureafter 16 hat 37 OCandclarified by centrifugation. Cells were washed once with PBS, harvested by scraping, and lysed in 1 mL of RIPA buffer (300 mM NaC1/100 mM Tris-HC1 containing 2% Triton X-100, 2% sodium deoxycholate, 0.2% SDS, 0.4% BSA, and 2 mM PMSF). Following an incubation of 30 min on ice, the lysate was clarified by centrifugation (30 min at 4000 rpm) and used immediately or was stored at -70 OC. %-Labeled proteins released into the conditioned media by the different cell lines were immunoprecipitated with 10 pg (specific or nonspecific) of antibody partially purified by 45% ammonium sulfate precipitation as previously described (Kessler, 1975). After solubilization, the immunoprecipitates were analyzed by 15% SDS-PAGE (Laemmli, 1970) and subsequent fluorography. Prestained molecular weight markers (Bio-Rad, Richmond, CA) were run in parallel lanes. Tunicamycin Treatment. Tunicamycin (Sigma, St. Louis, MO) was dissolved in 50 mM sodium carbonate (pH 10.0) and filter-sterilized with a 0.22-pm filter. Confluent monolayers of MDA-MB-231, MCF-7, and Hs578T cells were grown in IMEM in the presence of 20 pg/mL tunicamycin (unless otherwise specified) (Bringman et al., 1987) for 4 h prior to metabolic labeling. Metabolic labeling was then performed as described previously. Samples containingTGFa-like activitywere incubated with 20 pg of porcine pancreatic elastase (Sigma) dissolved in 50 mM glycylglycine, pH 7.9, for 1 h at 22 OC. The samples were then subjected to immunoprecipitationand SDS-PAGE analysis. Polyclonal and Monoclonal Antibodies. ( A ) Polyclonal Antibodies. Antiserum against human TGFa was obtained by immunization of a rabbit on day 0 with 400 fig of recombinant TGFa synthesized in Escherichia coli, kindly provided by R. Derynck, Genentech Corp. (Derynck et al., 1984). The immunogen was first conjugated to keyhole limpet hemocyanin (KLH) (Sutcliffeet al., 1980) and was emulsified in complete Freund’s adjuvant and was injected intrader-
7332 Biochemistry, Vol. 31, No. 32, 1992 mally at multiple sites. Additional injections were given as follows: day 60,175 p g of TGFa; days 90,150,180, and 210, 100 pg of TGFa. The booster injections were given subcutaneously at multiple sites in incomplete Freund's adjuvant. The rabbit serum was assayed for antibody titer by ELISA at 10-14 days following each injection. The antiserum collected at day 180, designated R399, was used for immunoprecipitation and radioimmunoassay. ( B ) Monoclonal Antibodies. A monoclonal antibody against recombinant TGFa was provided by R. Derynck (Derynck et al., 1984). Measurement of Anti- TGFa Antibody (R399) Levels by ELISA. Micro-Elisa plates (Dynatech-Immunolon 11, Dynatech Laboratories, Inc., Chantilly, VA) were coated for 16 h at 4 OC with 500 ng/mL recombinant TGFa in 50 mM sodium carbonate buffer (pH 9.6). The samples to be assayed (antibody) were serially diluted 1:lOOO-1:64 000 with 0.15 M NaCl, 0.05 M Tris-HC1 (pH 7.4), 2 mM EDTA, 5 mg/mL bovine serum albumin, and 0.05% Tween 20 (TBS-BSATween) and were incubated in the wells for 2 h at 37 OC. The plates were washed 5 times with PBS-Tween and then incubated for 1 h at 37 OC with horseradish peroxidaseconjugated goat anti-rabbit immunoglobulin in TBS-BSATween. The plates were then washed 5 times with PBSTween and incubated for 4 h at 22 OC with 100 pL per well of 0.1 mg/mL o-phenylenediamine/0.012% H202 in 0.1 M phosphate/citrate buffer (pH 5.0). The reaction was stopped by the addition of 50 pL/well of 2.5 N HzS04, and the absorbance was measured at 492 nm using a UR 700 microplate reader (Dynatech Laboratories). Radioimmunoassay (RIA): TGFa RIA. The presence of peptides immunologically related to TGFa was determined using a RIA kit with a polyclonal anti-rat TGFa and rat [ lZSI]TGFa(Biotope, Inc., Seattle, WA). This antibody does not cross-react with human EGF. Aliquots of conditioned media were reduced with 40 mM dithiothreitol and denatured by immersion for 1 min in a boiling water bath. Assays were done in duplicate according to the manufacturer's protocol, and each collection of conditioned media was assayed at least twice. N-Glycanase Digestion. The purified gp30 was subjected to digestion with N-glycanase. Samples equivalent to 100 ng were incubated with 50 pL of 0.2 M sodium phosphate (pH 8.6)/1.25% NP40, and 2-6 pg of N-glycanase (Genzyme Corp., Boston, MA) was subsequently added to each sample and incubated at 37 OC for 16 h; 50 pL of 3-fold-concentrated loading buffer was added before electrophoretic analysis, performed as outlined above. The gel was silver-stained. EGF Radioreceptor Assay. A43 1 membranes were prepared according to the method of Kimball and Warner (1984). A431 cells were disrupted under nitrogen and the nuclei and organelles pelleted by low-speed centrifugation. The membranes were then pelleted by centrifugation at 35 000 rpm for 1 h and resuspended in 20 mM HEPES buffer, pH 7.4. Membranes (2.5 pg/mL) were plated into 96-well plates and allowed todry overnightat 37 O C before use. Standard binding competition studies were performed using [1251] EGF (ICN, Costa Mesa, CA; specific activity 100 pCi/pg, about 50 000 cpm/well). A standard curve was constructed with 0.075-10 ng of unlabeled hEGF (receptor-grade, Collaborative Research). The different fractions to be analyzed were lyophilized and reconstituted in PBS (0.5 mL/500 mL of conditioned media). After incubation of the labeled EGF and 10 pL of the samples for 2 h at 37 OC in 200 pL of binding buffer (IMEM containing 50 mM HEPES and 0.1% BSA, pH 7.7),
Lupu et al. the wells were washed, cut from the plate, and counted. EGFcompeting activity was computed using a Hewlett Packard RIA program (Aitken et al., 1977). Anchorage-Independent Growth Assay. Soft agar cloning assays were carried out as previously described (Bates et al., 1988) using a 1-mL bottom layer of IMEM containing 0.6% Bacto-agar (Difco, Detroit, MI), 10% FBS, and 2 mM glutamine in 35-mm tissue dishes (Costar, Cambridge, MA). A 0.8-mL top layer of IMEM containing the test samples, 0.36% agar, 10% FBS, and 3 X lo4 NRK cells was added after solidification of the bottom layer. Each sample was plated in triplicate. All samples were sterilized by filtration using a 0.22-pm Millex CU Millipore filter before plating. Plates wereincubated in a humidified, 5% C02 atmosphere at 37 OC and were counted after 12-days incubation with a Bausch and Lomb stem cell colony counter (Artex Systems Corp., Farmingdale, NY). Anchorage-Dependent Growth Assay. NRK and 184NIA4T cells were grown in IMEM containing 5% FCS. Upon confluence, cells were detached using trypsin-versene (Biofluids, Rockville, MD) and passed at 1:20-1:50 dilutions. Cells were seeded in 12-well plates at 4000-10 000 cells/well, depending on the cell type (MDA-MB-23 1,8000 cells/well in serum-free IMEM). After 24 h, the medium was changed, and the cells were treated with EGF, TGFa, or gp30 harvested at 1,2, and 4 days, respectively, using trypsin-versene. The cells were counted using a Coulter counter. Heparin Affinity Chromatography. Media conditioned by MDA-MB-23 1 cells were clarified by centrifugation for 20 min at 2000 rpm at 4 OC. The supernatant was collected and stored at -70 OC. After the heparinaepharose (Pharmacia) was allowed to expand in PBS, 2 mL of gel was loaded on an Econo column (Bio-Rad) and washed with about 100 bead volumes of PBS. Conditioned media were run through the beads by gravity (flow rate 20-50 mL/h). The gel was then washed with 5 volumes of PBS and eluted stepwise with an increasing gradient of NaCl in 10 mM Tris-HC1, pH 7.0 (elution buffer). Gradient steps of 0.4 to 3.0 M NaCl were used in the elution buffer until the 280-nm absorption during each step returned to base line (usually 3-5 column bed volumes). The eluate was desalted on G-25 columns (Pharmacia) and filter-sterilized before use in the different bioassays. Pooled fractions containing active materials were also desalted on PDlO columns (Pharmacia) before running through HPLC and FPLC. Reversed-Phase High-pressure Liquid Chromatography (HPLC). ( A ) Steep Acetonitrile Gradient. Steep acetonitrile gradient and all other HPLC steps were carried out at room temperature after equilibration of the C3 reversed-phase column with 0.05% TFA (trifluoroacetic acid) in water (HPLC-grade). The sampleswere loaded and fractions eluted with a linear gradient ( 0 4 5 % acetonitrile in 0.05%TFA) at a flow rate of 1 mL/min over a 30-min period. Absorbance was monitored at 280 nm. One-milliliter fractions were collected and lyophilized before analysis for EGF receptorcompeting activity. ( B ) Shallow Acetonitrile Gradient. The pool of active fractions from the previous HPLC step was rechromatographed over the same column. Elution was performed with a 0-1876 acetonitrile gradient in 0.05% TFA over a 5-min period followed by a linear 1 8 4 5 % acetonitrile gradient in 0.05% TFA over a 30-min period. The flow rate was 1.0 mL/min, and 1-mL fractions were collected. Human TGFalike factor was eluted at a 30-32% acetonitrile concentration as a single peak detectable by RRA.
Biochemistry, Vola 31, No. 32, 1992 7333
TGFa-like Protein: Purification and Characterization Electrophoretic Elution of Radiolabeled Proteinfrom Gels. After fluorography of an SDS-PAGE, bands of interest were excised, and the protein was eluted by electrophoresis into dialysis tubing over 16 h at 120 V. The contents of the dialysis bag were cooled at 4 OC and then precipitated by the addition of trichloroacetic acid to a final concentration of 20%. The precipitateswere pelleted by centrifugation,washed twice with ethyl ether, and resuspended in loading buffer. Digestion Procedure for Purified Eluted Proteins. Electroeluted proteins were dissolved at approximately 0.5 mg/ mL in loading buffer which contained 0.125 M Tris-HC1(pH 6.8),0.5% SDS, lO%glycerol,andO.OOl% Bromophenol Blue. The samples were then heated at 100 OC for 5 min. Proteolytic digestion was carried out at 37 OC for 30 min by the addition of Staphylococcus aureus V8 protease (Sigma) to a final concentration of 25 pg/mL according to published methods (Cleveland et al., 1977). 8-Mercaptoethanol and SDS were subsequently added to final concentrations of 20% and 2%, respectively. Proteolysis was stopped by boiling for 2 min. The samples were then injected on a C18 reversedphase HPLC column (see above). Phosphorylationof the EGFReceptor. Subconfluent A43 1 cells were cultured in IMEM for 10-12 h. The cells were treated with 10-30 nM TGFa, EGF, or gp30 for 30 min at 37 OC. Cells were lysed in 20 mM Tris-HC1 (pH 7.4), 150 mM NaCl, 1% NP40, 1 mM EDTA, 2 mM PMSF, and 42 mM leupeptin and immunoprecipitated as described above using monoclonal antibody 225 directed against the EGF receptor (Oncogene Science, Manhasset, NY). The immunoprecipitates were washed 3 times with RIPA buffer and resuspended in 40 pL of TNE (0.01 M Tris-HC1, pH 7.5,O. 15 M NaCl, and 1 mM EDTA). Five microcuries of [-p32P]ATP was added to the immunoprecipitates,and the total ATP concentration was adjusted to 15 mM (final) in a volume of 60 pL. The reaction mixture was incubated for 5 min on ice before addition of 20 pL of 3X sample buffer. The samples were boiled for 5 min and analyzed by denaturing 7.5% SDSPAGE. R N A Extraction. Total cellular RNA was extracted from cells by homogenizing in guanidine isothiocyanate followed by centrifugation over a cesium chloride cushion. Poly(A)+ mRNA was eluted in 10 mM Tris after total cellular RNA was passed over an oligo(dt)-cellulose column (Pharmacia) equilibrated with 10 mM Tris/O.5 M NaCl, pH 8.0. After precipitationin ethanol (66%v/v) and 0.1 M acetic acid, both total and poly(A)+-selected RNAs were resuspended in 10 M Tris/ 1 mM EDTA buffer and separated on 1% agarose/6% formaldehyde gels. Electrophoresis was carried out at 20 V over 14-16 h in 5 mM NaOAc, 1 mM EDTA, and 20 mM 3-(N-morpholino)propanesulfonic acid, pH 7.0 (MOPS, Sigma). The gels were stained with 2.0 g/mL ethidium bromide to allow inspection of the quality and quantity of RNA. In vitro translation assays were performed using wheat germ kit according to the manufacturer's instructions (Promega).
RESULTS Identification of a TGFa-like Polypeptide in MDA-MB231 Human Breast Cancer Cells. Earlier experiments using gel filtration chromatographysuggested the presence of a gp30 TGFa-like related peptide in conditioned media from MDAMB-231 cells. To determine whether this protein was recognized by antibodies developed against mature 6-kDa TGFa, MDA-MB-231 cells were metabolically labeled with [%]methionine and [35S]cysteine. Metabolically labeled
;20] 0
0-0 0-0
a-
A
MDA- ME 231 H 8 HS 5781
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0 101 c
In m
n
o
fLA-n-n---~-~---~ 1
10
100
35S-protein added ( cpm x l o 3 )
FIGURE 1: TGFa radioimmunoassay of metabolically labeled conditionedmediafrom MDA-MB-231, Hs578T, andH8 cells. Wells of 96-well plates were coated with 10 Hg/mL polyclonal antibody R399 for 2 h at 37 "C. Free binding sites were blocked with 1.0% BSA (RIA-grade)for 1 hat 22 "C.Serial dilutions of metabolically labeled conditioned media were added and incubated for 2 h at 37 "C.After a wash with 0.1%Tween 20 in PBS, radioactivity retained in the wells was counted. Data points represent the mean of duplicate values. conditioned media from MDA-MB-231, TGFa-transfected MCF-7 (HS), and HS578T cells were tested by solid-phase RIA for immunoreactivitywith a polyclonal antibody (R399) and a monoclonal antibody raised against recombinant 6kDa TGFa. Metabolically labeled TGFa-like material from MDA-MB-23 1 cells reacted only with the polyclonal antibody. In contrast, both the polyclonal and the monoclonal antibodies cross-reactedwith metabolically labeled material derived from H8 cells. No immunoreactivity was observed when preimmune serum was used (NRS or NMS). Furthermore, no cross-reactivity was observed when metabolically labeled conditioned medium from HS578T breast carcinosarcoma cells was used (Figure 1). HS578T cells do not produce TGFa mRNA (Bates et al., 1988). Specificity of the assay was demonstrated using a competition RIA with unlabeled recombinant TGFa (data not shown). Labeled material from MDA-MB-23 1, H8, and Rat-FeSrV cells was immunoprecipitated with the anti-TGFa polyclonal antibody. Detection of an immunoreactive species of approximately gp30 size verified the secretion of a high molecular weight TGFa-like polypeptide in MDA-MB-23l cells. H8 cells, which overexpress classical TGFa, yielded a 6-kDa product. The expected 18-kDa precursor of the classical 6kDa TGFa was precipitated from Rat-FeSrV cells, which are known to secrete the TGFa precursor. The intensity of the bands diminished when the immunoprecipitation was performed in the presence of excess unlabeled TGFa (Figure 2). None of these bands were immunoprecipitated by preimmune rabbit serum. Deglycosylation and Elastase Cleavage of the Larger TGFa-like Polypeptide. The apparent heterogeneity in size of the authentic TGFa species described in the literature and its potential for N-linked glycosylation at Asn-25 (Bringman et al., 1987) led us to question whether the 30-kDa TGFa-like polypeptide secreted from MDA-MB-231 cells was a glycosylated form of TGFa. When MDA-MB-231 cells were incubated with tunicamycin, an inhibitor of cotranslational N-linked glycosylation, and the media were immunoprecipitated with the anti-TGFa polyclonal antibody, a species of 22 kDa substituted for that previously observed at 30 kDa (Figure 3A). Tunicamycin treatment did not significantly affect the levels of secreted TGFa activity as determined by both RIA and EGF receptor binding assays (data not shown).
Lupu et al.
7334 Biochemistry, Vol. 31, No. 32, 1992 Hs578T I
1
MDA-MB231
2 3 "4
5
6"7
H8 8
Rat-FeSrV Q " 1 0 11 12'
kDa
30 I)
18 I)
FIGURE2: Immunoprecipitationof TGFa speciesfrom metabolically labeled media of MDA-MB-231, H8, and Rat-FeSrV cells. Immunoprecipitation was carried out with polyclonal antibody R399 or with preimmuneserum (normal rabbit serum, NRS). The precipitates were analyzed by 15%SDS-PAGE under reducing conditions and fluorographed. Hs578T (lanes 2 and 3), MDA-MB-231 (lanes 5 and 6), H8 (lanes 8 and 9), and Rat-FeSrV (lanes 1 1 and 12) media were precipitatedwith polyclonal antibody R399 or preimmuneserum NRS (lanes 1,4,7, and 10). Precipitationwas also performed in the presence of 500 ng of recombinant TGFa (lanes 3, 6, 9, and 12). Molecular masses are indicated by arrows (kDa, kilodaltons). We next addressed whether this deglycosylated species was a precursor form of mature TGFa. The 22-kDa polypeptide was treated with elastase, an enzyme known to cleave off 6-kDa mature TGFa from several TGFa precursors of different sizes (Luetteke et al., 1988;Madtes et al., 1988). An ll-kDa product was obtained (Figure 3B). This was in contrast to the 6-kDa product that was obtained when the deglycosylated 16-kDa TGFa precursor secreted by RatFeSrV cells was treated with elastase (Figure 3B). Shorter exposure of the gel showed clearly a precipitated band near the 11-kDa molecular mass. Furthermore,when purified gp30 was treated with N-glycanase,a 22-kDa product was detected by silver staining (Figure 3C). The biological activity of the purified gp30 was determined by its ability to stimulate NRK colony formation and EGF receptor phosphorylation, as described in Figures 9 and 10. The absence of cleavageof the purified gp30 after 0-glycanase treatment (data not shown) suggests that no O-glycosylation occurs in this system. Purification of the TGFa-like Polypeptide. TGFa-like material was isolated from serum-free conditioned media of MDA-MB-231 cells. Levels of TGFa-like polypeptide were quantified by three independent assays: capacity to induce anchorage-independent growth of NRK fibroblasts in soft agar; ability to compete with [*251]EGFfor EGF receptor binding on A43 1 human carcinomacell membranes;and crossreactivity with polyclonal antibodies raised against mature TGFa. To determine the approximate molecular weight of the MDA-MB-231-derived TGFa-like polypeptide, 5 mL of 100-fold-concentrated,dialyzed conditionedmedium was chromatographerl by gel filtration using Sephadex G-100. Elution was performed with 1.O M acetic acid, and fractions were characterized for protein content. TGFa-like activities were eluted from the column in a single broad peak. The relative amounts of receptor binding activity and immunoreactivity present in these fractions, however, appeared to differ. Further analysis of the TGFa-like polypeptide from MDA-MB-23 1 cells was carried out using heparin-Sepharose affinity chromatography. In all experiments, less than 20% of the TGFa activity loaded onto the column was recovered in the unabsorbed fractions. A sharp peak of EGF receptor binding
activity was eluted by heparinsepharose chromatography at a concentration of 0.4-0.6 M NaCl (Figure 4A). The major activity which retained 70-8096 of the loaded activity is shown in Figure 4B. In contrast, neither EGF, TGFa, nor their precursors bind to heparin (data not shown). The heparin binding TGF-like polypeptide (HP-TGFa-like) was further purified by reversed-phase chromatography (HPLC) in two steps. A pool of fractions containingEGF receptor-competing activity from heparin-Sepharose chromatography was reconstituted in 0.05% TFA in water and then chromatographed on a pBondapak C3 column. A steep acetonitrile gradient (0-100%) was used in this step, The elution pattern is shown in Figure 5 (top). The TGFa-like polypeptide elutes as a sharp peak in 30% acetonitrile and is separated from the bulk of the contaminating proteins. The capacity of the individual fractions to compete for EGF receptor binding and to stimulate the growth of NRK cells in soft agar was determined (Figure 5, bottom). A pool of the active fractions (indicated with an horizontal bar) was rechromatographed on the same column. Fractions were eluted with a 0-20% acetonitrile gradient in 0.05% TFA for 5 min followed by a linear 2040% acetonitrile gradient. The TGFa-like polypeptide activity was eluted at 25-30% acetonitrile and effectively separated from other contaminant proteins. We used size-exclusion chromatography under acidic conditionsto verify the size of the TGFalike peptide. The active fractions for EGF receptor-competing activity (Figure 6A) were approximately 30 kDa in size. The purified 30-kDa TGFa-like polypeptidewas analyzed by SDSPAGE. An exampleof the apparent homogeneity of the active material is shown in Figure 6B [see also Lupu et al. (1991)l. At this point, we cannot conclude that this purified polypeptide precisely comigrates with the 35Smetabolically labeled material described earlier, although both migrate at approximately 30 kDa. In other experiments, when traces of metabolically labeled MDA-MB-231-conditioned media were used as a tracer, comigration of 35S-labeledmaterial with 30-kDa protein was observed (data not shown). A summary of the steps leading to the isolation and purification of TGFa-like polypeptide is presented in Table I. A 27% recovery of activity and approximate 5400-fold purification were achieved. Biological Characterization of the TGFa-like Material. The EGF receptor binding activity of the gp30 TGFa-like protein was compared with that of EGF in a radioreceptor assay. Bothgrowth factors competed with [1251]EGFforEGF receptor sites on A431 membranes. The specific EGFcompetingactivity of the purified TGFa-like polypeptide was found to be (1-1.5) X lo6 units/mg; 1.1 ng/mL TGFa-like polypeptide was required to inhibit EGF binding by 50%. TGFa-like polypeptide was as effective as EGF in receptor binding. Furthermore, the purified gp30 TGFa-like polypeptide stimulated the growth of serum-deprived NRK fibroblasts (Figure 7A) and inducedcolony formation of these cells in soft agar (Figure 7B). The bioactivity of the purified TGFa-like polypeptide was also tested by anchorage-dependent growth assays of the carcinogen-immortalized human mammary epithelial cells 184AlN4 (data not shown) and anchorage-independent growth assays of 184A 1N4-derived cells partially transformed by SV40 T antigen, 184AlN4T (data not shown). The biological activity of the purified gp30 TGFa-like factor was further assessed by examiningits ability to induce autophosphorylation of the EGF receptor. A43 1 cells, which overexpress the EGF receptor, were incubated with various concentrations of EGF, TGFa, or HB-TGFalike growth factors. Each of the three peptides similarly
Biochemistry, Vol. 31, No. 32, 1992 7335
TGFa-like Protein: Purification and Characterization
C
B
A kDa
kDa
3022
18 16-
+
4
116.3-
I1
21 ( 3 4 1
+TUN
-TUN
kD8
kDa
1 5 %7
1
81
1
2
3
4
5
6
7
1
3
2
4
5
-TUN +TUN
FIGURE 3: Inhibition of glycosylation, enzymatic deglycosylation, and specific cleavage of the TGFa-related species. (A) Metabolic labeling of MDA-MB-231 (lanes 1-4) and Rat-FeSrV (lanes 5-8) was carried out in the absence or presence of tunicamycin (20 pg/mL). Immunoprecipitations were performed with polyclonal antibody R399 (lanes 1, 3, 5, and 8) or preimmune serum (lanes 2,4,6, and 7) followed by SDS-PAGE analysis. (B) Metabolic labeling of conditioned media derived from MDA-MB-231 and FeSrV cells was carried out in the presence of tunicamycin as described above, followed by specific cleavage with 25 pg of elastase. Immunoprecipitation was then performed with R399 antibody (lanes 1,5, and 6) and NRS (lanes 2,3, and 7). Lanes 1 and 2 represent precipitated material derived from MDA-MB-231 cells, lanes 5-7 represent precipitated material derived from FeSrV cells, and lane 4 represents immunoprecipitationof mature TGFa. Samples were analyzed by 15% SDS-PAGE. Molecular mass markers are indicated by arrows (kDa, kilodaltons). (C) Samples equivalent to 100 ng of purified gp30 derived from MDA-MB-231 cells (lane 1) were incubated as described under Materials and Methods with buffer alone (lane 2) or with 2 (lane 3), 4 (lane 4), and 6 pg (lane 5) of N-glycanase followed by silver staining of a 15% SDS-PAGE.
B
A e
2000 1
I
0
-E 2a
Protein
RRA
kd
CI 1
LL
c3
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D
1500
I D
-
n
v
1
67-
26-
1000
3 0
-
0
1
14-
500 -
( -
100
.C
0
+
2
K
(2:
Q
07 fraction number
FIGURE 4: HeparinSepharose affinity chromatography of TGFa-like activity from MDA-MB 23 1 cells. (A) Affinity chromatography of 1 L of unconcentrated conditioned media was performed on a heparin-sepharose column with elution by a steep gradient from 0.4 to 3.0 M NaCl. Absorbance was monitored at 280 nm (open triangles). Three-milliliter fractions were collected and lyophilized after desalting. The lyophilized fractions were reconstituted in 500 pL. Aliquots from the resulting 500-pL fractions were analyzed for EGF receptor binding activity (closed circles). The horizontal bar indicates the fractions pooled for reverse-phaseC3 chromatography (see Figure 6). (B) An aliquot of 30 ng from the active fraction was analyzed by 15% SDS-PAGE followed by silver staining. Lane 1 represents unconcentrated media and lane 2 the active fraction after chromatorgraphy.
stimulated phosphorylation of the EGF receptor (Figure 8). In Vitro Translations. In order to determine if HB-TGFalike is a product different than a posttranslational modification of the TGFa precursor, we performed in vitro translations of mRNA isolated from MDA-MB-231 cells. As a control, mRNA derived from H8 cells was used. Immunoprecipitation of the specific translated products was performed after treatment with elastase. The precipitated 22-kDa polypeptide for MDA-MB-231 mRNA (Figure 9A, lane 1) and 16kDa polypeptide for H8 mRNA (Figure 9A, lane 7) were subjectedto elastase cleavage,resulting in peptides of 11 kDa for MDA-MB-231 cells (Figure 9A, lane 3, and Figure 9B, lane B) and 6 kDa for H8 cells (Figure 9A, lane 11, and Figure 9B, lane A). These in vitro translated proteins corresponded to the sizes observed earlier for the deglycosylated 22-kDa polypeptide from MDA-MB-23 1 cells and the unglycosylated 16-kDapolypeptide from Rat-FeSrV cells.
The in vitro translated HB-TGF-likeproducts are summarized in Table IIB. These results indicated that the 11-kDa peptide derived from MDA-MB-23 1 cells is probably not a result of posttranslational modifications. Peptide Mapping. In order to determine the degree of homology between gp30 and mature TGFa, peptide mapping was performed using the method of Cleveland. Immunoprecipitation of metabolicallylabeled conditioned media from MDA-MB-231, H8, and Rat-FeSrV cells was carried out with the R399 anti-TGFa polyclonal antibody. Precipitates were analyzed by SDS-PAGE, and the specific bands were electroeluted (gp30 from MDA-MB-231 cells, 6 kDa from H8 cells, and 18 kDa from Rat-FeSrV cells). These proteins were subjected to enzymatic treatment with N-glycanase and elastase. The sizes of the precipitated bands are summarized in Table IIA. The products were then subjected to peptide digestion using 25 pg/mL V8 protease. After complete
Lupu et al.
7336 Biochemistry, Vol. 31, No. 32, I992 1 .o
FeSrV cells were essential identical. Similar results were obtained with 40 p g of V8 protease, indicating that the concentration of the enzyme was not responsible for the differential peptide cleavage. To further explore the identity of the two apparently distinct protein products, V8 protease digestion was performed on electroeluted polypeptides after immunoprecipitations of in vitro translated products. The resulting peptides were analyzed by C18 reversed-phase chromatography under identical acetonitrile gradients. Peptide mapping of the translated products had a very similar profile as the purified gp30 factor after treatment with N-glycanase and elastase as shown in Table 111. These experiments were done 3 times for each V8 protease concentration, and the results were reproducible.
0.8
f
0.6
t
e
s 4
DISCUSSION
500 400 \
B
Y
300
I k.
2
200
4
a a
100
16
13
25
22
19
20
retention lime (min)
FIGURE 5: Reversed-phaseCs chromatographyof conditioned media from MDA-MB-231 cells after heparin affinity chromatography. A sampleof 1.5 mg of total protein from the active fraction eluted from heparinsepharose was loaded on a pBondapak Cj column in 0.05% TFA. Elution was achieved with a linear gradient of acetonitrile (&loo%). As described under Materials and Methods, 1 mL/min fractions were collected, lyophilized,and reconstituted in 200 pL of HzO. Theabsorbanceat &-is indicatedin the top panel. Fractions were assayed for EGF-competing activity (closed circles) using a 100-pLaliquot or for NRK colony formation (open circles) using a 50-pL aliquot. 0
4001
A
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0
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20
25
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30
fraction number
FIGURE 6: Size-exclusionrechromatographyof TGFa-likematerial. (A) Pooled fractions from the previousstepwere rechromatographed by acidic size-exclusion chromatography (Suprose 12, Pharmacia, NJ) toverify thesizeof thegrowth factor. Theelution was performed with 1 M acetic acid; 1-mL fractions were collected and then lyophilized. After reconstitution with 100 p L of Tris-HC1 (pH 7 . 9 , the fractions were analyzed for EGF-competingactivity by radioreceptor assay. (B) Homogeneity of proteins contained in the pooled fractions from the final purification step were determined by silver staining of a 15%SDS-PAGE. This reproduces Figure 3C, lane 2. digestion, the samples were analyzed by C18 reversed-phase chromatography. Three major peptide peaks for each cell line eluted at different acetonitrile concentrations by reversedphase chromatography. However, theconcentrations at which the peptides isolated from MDA-MB-231 cells eluted were different from the peptides isolated from H8 and FeSrV cells as described in Table I11 and Figure 10. The peptide elution patterns of the 6-kDa TGFa derived from H8 cells and Rat-
GeneralSignificance. On the basis of in vitro studies, TGFa has been postulated to be a autocrinemediator of tumor growth (Sporn et al., 1980). Studies suggest that TGFa activity can be detected in body fluids of cancer patients. Its presence may provide important information concerning the biology of a patient's tumor (Stromberg et al., 1986a,b; Twardzick et al., 1982; Sherwin et al., 1983). In effusions from breast cancer patients, the presence of TGFa was correlated with the absence of estrogen and progesterone receptors in primary breast tumors (Artega et al., 1988). We have shown that hybridization of MDA-MB-231 mRNA to a TGFa cDNA probe yielded an mRNA of the predicted size of 4.8 kb as well as a novel 1.6-kb species (Bates et al., 1988). The significance of the 1.6-kb mRNA is unknown, but it was also seen in poly(A)+-enriched RNA from human primary breast cancers. Expression of the 4.8- and 1.6-kb transcripts in MDA-MB231 cells is associated with the presence of the high molecular weight form of TGFa secreted by MDA-MB-231 cells as detected by RIA using antibodies against mature TGFa. However, the presence of the 1.6-kb transcript is not in all cases associated with the 30-kDa polypeptide (Susan Bates, personal communication). The extent of production of this TGFa-like polypeptide in breast cancer cell lines remains to be seen. In the present study, we have shown that a heparin binding TGFa-like growth factor is produced by the estrogen receptornegative human breast adenocarcinoma cell line MDA-MB231 and is released into the media. We purified this polypeptide to apparent homogeneity and partially characterized it. Glycosylation and Secretion. Multiple polypeptide species with TGFa-like activity have been described with sizes ranging from 6 to 68 kDa. These forms are most likely processing products derived from a common glycosylated and palmitoylated precursor. It has been suggested that the 18-kDa species corresponds to the entire TGFa precursor while the 6-kDa and larger forms represent N-glycosylated forms of TGFa released after proteolytic cleavage of this same transmembrane TGFa precursor. None of these species has a size similar to the gp30 with TGFa-like activities describe in this study. Evidence for N-glycosylation of the 22-kDa TGFalike precursor form derived from MDA-MB-23 1cells is shown by a reduction in molecular weight after tunicamycin treatment. Likewise, N-glycanase treatment of the purified gp30 yielded a protein of the same molecular weight. The presence of tunicamycin had little effect on the secretion of gp30 into the medium. It is therefore unlikely that N-glycosylation plays an important role in secretion or cell-surface transport as observed for other membrane-anchored forms of growth factors.
Biochemistry, Vol. 31, No. 32, 1992 1331
TGFa-like Protein: Purification and Characterization
Table I: Purification of TGFa-like Activity from Conditioned Medium from MDA-MB-231 Cellse
0-oeontrd 0-0 + E V 10 d m l
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A-A
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Y
2 3 -
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